Vaccination with antigenic peptides that stimulate a melanoma-specific cytotoxic T lymphocyte (CTL) response is a promising approach to the treatment of melanoma. The work proposed here is predicated on the hypothesis that T cell-mediated immunotherapy will be most successful when: (i) multiple peptide epitopes derived from different source proteins are presented by the same MHC molecule: and (ii) peptide epitopes are presented on multiple MHC molecules within the same individual. Immunization with peptides derived from multiple source proteins rather than a single source protein should minimize the probability that a gene mutation or deletion will result in immune escape, while targeting peptide epitopes to multiple class I MHC molecules should minimize the likelihood that mutation or loss of a single class I gene will result in immune escape, and at the same time broaden the percentage of the population that can receive treatment. Therefore, the overall objective of the research proposed here is to expand the available repertoire of melanoma-derived peptide antigens, both with respect to their absolute number and the range of MHC molecules on which they can be presented, with the ultimate goal (although beyond the scope of this proposal) of using the peptides as a vaccine for the treatment of melanoma. The specific aims of this research proposal are to: (1) determine if the utility of the known class I MHC-restricted melanoma epitopes can be expanded by first determining if they are naturally processed and presented by other members of the same class I MHC supertype family, and secondly by asking if the complexes are capable of stimulating a melanoma-specific CTL response; (2) utilize SRM scanning mass spectrometry to identify new CTL epitopes derived from gp 100, tyrosinase, MART-1, TRP- 1, TRP-2, MC1R, MAGE-3, and MAGE-12; (3) determine the extent to which the gplOO, tyrosinase, MART-l, TRP-1, TRP-2, MC1R, MAGE-3, and MAGE-12 melanoma antigens give rise to HLA-A1, A2, A3, B7, or B8 MHC associated epitopes that can be recognized by melanoma-specific CTL; and (4) identify and characterize novel epitopes recognized by melanoma-specific CTL. The methods employed in this proposal will involve a unique combination of cellular immunology and mass spectrometry which will allow for the identification of antigenic peptides both with and without pre-existing CTL. The successful completion of this research should impact on melanoma therapy by increasing the number of available antigens for vaccination, as well as by increasing the percentage of the population that can avail themselves of this form of treatment.